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PubMedDate not supplied

Reduced immunogenicity of beta-lactoglobulin by conjugation with carboxymethyl dextran.

We prepared two beta-lactoglobulin (beta-LG)-carboxymethyl dextran (CMD) conjugates (Conj. 10A and Conj. 10B) by using a water-soluble carbodiimide to decrease the immunogenicity of beta-LG. The molar ratios of beta-LG to CMD in the conjugates were 5:1 (Conj. 10A) and 2:1 (Conj. 10B). The beta-LG-CMD conjugates maintained the retinol-binding activity of native beta-LG. Intrinsic fluorescence study indicated that shielding of the surface of beta-LG by CMD occurred in each conjugate, which was eminent in Conj. 10B. A local conformational change around (125)Thr-(135)Lys (alpha-helix) in each conjugate was detected by ELISA with monoclonal antibodies. The denaturation temperature of beta-LG evaluated by differential scanning calorimetry was greatly enhanced in each conjugate. The anti-beta-LG antibody response was markedly reduced after immunization with the beta-LG-CMD conjugates in BALB/c, C57BL/6, and C3H/He mice. We determined the B cell epitopes of beta-LG and each conjugate recognized in these mice and found that the linear epitope profiles of the beta-LG-CMD conjugates were similar to those of beta-LG, while the antibody response for each epitope was dramatically reduced. The reduced immunogenicity of beta-LG was most marked in the case of Conj. 10B, which contained more CMD than Conj. 10A, and was effectively shielded by CMD. We concluded that masking of epitopes by CMD is responsible for the decreased immunogenicity of the beta-LG in these conjugates.

Animals↗
PubMed2002

Does beta-lactoglobulin denaturation occur via an intermediate state?

The denaturation of beta-lactoglobulin (BLG) in the presence of urea and GuHCl has been investigated at different pH values with various spectroscopic techniques. The equilibrium denaturation free energy values, obtained by linearly extrapolating the data to vanishing denaturant (DeltaG(D)(H2O)), are compared and discussed. The fit of the spectroscopic data monitoring the denaturation of BLG has been approached, at first, with a two-state model that describes the protein transition from the folded state (at each pH and in the absence of denaturant) to the denatured state, but in particular, along the GuHCl denaturation pathway some evidence is found of the presence of an intermediate state. Time-resolved fluorescence experiments performed on the BLG-ANS (1-anilino-8-naphthalenesulfonate) complex help to understand the results. Fluorescence polarization anisotropy (FPA) measurements accompanying the denaturation process show the presence of a fast rotational diffusion of the ANS probe, and the data are interpreted in terms of local fluctuations of a still structured tract of the denatured protein where the probe is bound.

Animals↗
PubMed2002

Peptide models of folding initiation sites of bovine beta-lactoglobulin: identification of nativelike hydrophobic interactions involving G and H strands.

In an attempt to characterize the early folding events in bovine beta-lactoglobulin (BLG), a set of peptides, covering the flexible N-terminal region and the stable C-terminus beta-core, was synthesized and analyzed by circular dichroism and by nuclear magnetic resonance in water, trifluoroethanol (TFE), and sodium dodecyl sulfate (SDS) below and above the critical micellar concentration. The role of local and long-range hydrophobic interactions in guiding the folding has been investigated. For the peptide fragment covering the more flexible N-terminal region of BLG (beta-strands A, B), where both theoretical predictions and kinetic refolding experiments suggested the formation of non-native alpha-helix, no native long-range contacts were identified, and a helical secondary structure was stabilized only in the presence of 25 mM SDS. At variance, in 50% (v/v) TFE, native, long-range hydrophobic interactions were observed in the peptide covering the core region comprising G and H beta-strands. The side chains involved in these interactions form a nativelike hydrophobic cluster, thus suggesting that the GH region may act as the folding initiation site for BLG. This result is reinforced by the identification, in the urea denaturated BLG, of residual structure located at the level of the GH interface, as evidenced by NMR analysis. These results, in excellent agreement with kinetic, thermodynamic, and cold denaturation folding data, once more underline the utmost importance of the GH region for the stability and folding of BLG. Severe aggregation effects prevented the structural analysis of the peptide covering the EFGH region, indicating that this larger segment does not represent an independent folding domain and that the terminal alpha-helix is necessary for stabilizing the BLG folding core.

Amino Acid Sequence↗
PubMed2002

pH and ionic strength dependence of protein (un)folding and ligand binding to bovine beta-lactoglobulins A and B.

Formation of complexes between bovine beta-lactoglobulins (BLG) and long-chain fatty acids (FAs), effect of complex formation on protein stability, and effects of pH and ionic strength on both complex formation and protein stability were investigated as a function of pH and ionic strength by electrophoretic techniques and NMR spectroscopy. The stability of BLG against unfolding is sharply affected by the pH of the medium: both A and B BLG variants are maximally stabilized against urea denaturation at acidic pH and against SDS denaturation at alkaline pH. The complexes of BLGB with oleic (OA) and palmitic acid (PA) appear more stable than the apoprotein at neutral pH whereas no differential behavior is observed in acidic and alkaline media. PA forms with BLG more stable complexes than OA. The difference between the denaturant concentration able to bring about protein unfolding in the holo versus the apo forms is larger for urea than for SDS treatment. This evidence disfavors the hypothesis of strong hydrophobic interactions being involved in complex formation. Conversely, a significant contribution to FA binding by ionic interactions is demonstrated by the effect of pH and of chloride ion concentration on the stoichiometry of FA.BLG complexes. At neutral pH in a low ionic strength buffer, one molecule of FA is bound per BLG monomer; this ratio decreases to ca. 0.5 per monomer in the presence of 200 mM NaCl. The polar heads of bound FA appear to be solvent accessible, and carboxyl resonances exhibit an NMR titration curve with an apparent pK(a) of 4.7(1).

Animals↗
PubMed2003

Trifluoroethanol-induced beta --> alpha transition in beta-lactoglobulin: hydration and cosolvent binding studied by 2H, 17O, and 19F magnetic relaxation dispersion.

Alcohols, such as 2,2,2-trifluoroethanol (TFE), have been shown to induce a cooperative transition to an open helical structure in many proteins, but the underlying molecular mechanism has not been identified. Here, we employ the technique of magnetic relaxation dispersion (MRD) to study the TFE-induced beta --> alpha transition of beta-lactoglobulin at pH 2.4. Unlike traditional techniques that focus on protein secondary structure, the MRD method directly monitors the solvent, providing quantitative information about preferential solvation and solvent penetration and about the overall size and structural integrity of the protein. In this multinuclear MRD study, we use the (2)H and (17)O resonances to examine hydration and the (19)F resonance to study TFE. The transformation from the native to the helical state via an intermediate state at 300 K is found to be accompanied by a progressive expansion of the protein and loss of specific long-lived hydration sites. The observation of (17)O and (19)F dispersions from the helical state shows that water and TFE penetrate the protein. The MRD data indicate a strong accumulation of TFE at the surface as well as in the interior of the protein. At 277 K, BLG is much less affected by TFE, remaining in the native state at 16% TFE, but adopting a nonnative structure at 30% TFE. This nonnative structure is not penetrated by long-lived water molecules. The implications of these findings for the mechanism of TFE-induced structural transformations are discussed.

Animals↗
PubMed2004

A recombinant C121S mutant of bovine beta-lactoglobulin is more susceptible to peptic digestion and to denaturation by reducing agents and heating.

The lipocalin beta-lactoglobulin (BLG) is the major whey protein of bovine milk and is homodimeric at physiological conditions. Each monomer contains two disulfide bonds and one cysteine at position 121 (C121). This free thiol plays an important role in the heat-induced aggregation of BLG and, possibly, in its conformational stability. We describe here the expression in the yeast Pichia pastoris of a mutant bovine BLG, in which C121 was changed into Ser (C121S). Circular dichroism and high-performance liquid chromatography experiments, together with the X-ray crystal structure, show that the C121S mutant retains a nativelike fold at both neutral and acid pH. The mutation completely blocks the irreversible aggregation induced by heat treatment at 90 degrees C. Compared to the recombinant wild-type protein, the mutant is less stable to temperature and disulfide reducing agents and is much more sensitive to peptic digestion. Moreover, its affinity for 1-anilino-8-naphthalenesulfonate is increased at neutral and acid pH. We suggest that the stability of the protein arising from the hydrophobic effect is reduced by the C121S mutation so that unfolded or partially unfolded states are more favored.

Anilino Naphthalenesulfonates↗
PubMed2006

Lipid-induced conformational transitions of beta-lactoglobulin.

Bovine beta-lactoglobulin (betaLG) provides an excellent model protein system for beta-to-alpha conformational change, but its behavior varies when the change is induced by alcohols, surfactants, or lipid vesicles. Here the interaction and orientation of betaLG in association with various artificial lipid vesicles at neutral and acidic pH have been studied by use of several complementary spectroscopic techniques. Circular dichroism (CD) and Fourier transform infrared (FTIR) spectra demonstrated that betaLG acquires a non-native alpha-helical structure upon binding with anionic lipids, while zwitterionic lipids do not have a significant effect on its conformation. The degree of induced alpha-helix depends on the lipid concentration and is strongly affected by the charge of the protein and lipids as well as the ionic strength of the solution. Near-UV CD and Trp emission spectra revealed that the tertiary structure of lipid-bound betaLG is highly expanded but not completely disrupted. Fluorescence quenching together with a Trp emission blue shift showed that the Trp residues remain largely shielded from the solvent when interacting with DMPG, which would be consistent with at least some portions of betaLG having been inserted into the lipid membrane. The orientations of the alpha-helix and beta-sheet axes in membrane-bound betaLG were found to be parallel and perpendicular, respectively, to the membrane film normal, as determined by use of polarized attenuated total reflection (ATR) FTIR spectra. Our findings reveal that the lipid-induced beta-to-alpha transition in betaLG, accompanied by a substantial disruption in tertiary structure, is mainly driven by strong electrostatic interactions. Once the tightly packed betaLG is disrupted, hydrophobic residues become exposed and available for insertion into the lipid bilayer, where hydrophobic interaction with the lipids may play a role in stabilizing the helical components.

Animals↗
PubMed1996

Infrared and circular dichroism spectroscopic characterization of structural differences between beta-lactoglobulin A and B.

Structural differences between two genetic variants of bovine beta-lactoglobulins (type A and B) in aqueous solutions were characterized using Fourier transform infrared and circular dichroism spectroscopies. To probe differences in structural dynamics, the effects hydrogen-deuterium exchange were also compared for the two proteins. The infrared spectra recorded in H2O solution for the two proteins were nearly identical in the conformationlly sensitive amide I region. The only exceptions were small differences at the band ascribed to a high-wavenumber beta-sheet component near 1693 cm-1 and the band assigned to turns at 1684 cm-1. In contrast, when the proteins were prepared in D2O solution, marked spectral differences were observed at all regions ascribed to beta-sheet and turn structures. These differences are consistent with the structural differences of the two variants at amino acid residues 64 and 118, which are located at a turn and a beta-sheet structure, respectively, as revealed by X-ray crystallographic studies [Monaco et al. (1987) J. Mol. Biol. 197, 695-706]. The circular dichroism spectra for the two proteins were essentially identical, both before and after hydrogen-deuterium exchange. Therefore, hydrogen-deuterium exchange did not alter the proteins' secondary structure. The enhancement of the amide I spectral difference upon hydrogen-deuterium exchange was ascribed to the differences in the structural mobility of the two proteins. Since the rate of exchange was greater for variant A, it was concluded that this variant has greater structural mobility than variant B. These findings indicate that the combination of infrared spectroscopy and hydrogen-deuterium exchange has great potential in characterization of even subtle structural differences in proteins induced by naturally occurring point mutations and/or site-directed mutagenesis.

Animals↗
PubMed1997

Fatty acids and retinoids bind independently and simultaneously to beta-lactoglobulin.

beta-Lactoglobulin (Big) binds 1 mol of a fatty acid spin-label analog, 5-doxylstearic acid (5-DSA), per mole of protein with a dissociation constant Kd = 0.8 microM for the strongest binding site. There are also several weaker sites for this ligand. Blg saturated with either retinol or retinoic acid binds 5-DSA with essentially equal affinity (Kd = 0.6 and 1 microM, respectively). Palmitic acid and SDS displace bound 5-DSA from Blg. However, unlike palmitic acid, 5-DSA binding does not enhance the structural stability of Blg to urea denaturation. The spin-labeled fatty acid also binds to the protein at low pH, presumably at secondary fatty acid binding sites. These results suggest that Blg binds at least two different types of hydrophobic ligands simultaneously.

Binding, Competitive↗
PubMed1998

Structural basis of the Tanford transition of bovine beta-lactoglobulin.

The structures of the trigonal crystal form of bovine beta-lactoglobulin variant A at pH 6.2, 7.1, and 8.2 have been determined by X-ray diffraction methods at a resolution of 2.56, 2. 24, and 2.49 A, respectively. The corresponding values for R (Rfree) are 0.192 (0.240), 0.234 (0.279), and 0.232 (0.277). The C and N termini as well as two disulfide bonds are clearly defined in these models. The glutamate side chain of residue 89 is buried at pH 6.2 and becomes exposed at pH 7.1 and 8.2. This conformational change, involving the loop 85-90, provides a structural basis for a variety of pH-dependent chemical, physical, and spectroscopic phenomena, collectively known as the Tanford transition.

Animals↗
PubMed1999

Differences between the pressure- and temperature-induced denaturation and aggregation of beta-lactoglobulin A, B, and AB monitored by FT-IR spectroscopy and small-angle X-ray scattering.

We examined the temperature- and pressure-induced unfolding and aggregation of beta-lactoglobulin (beta-Lg) and its genetic variants A and B up to temperatures of 90 degrees C in the pressure range from 1 bar to 10 kbar. To achieve information simultaneously on the secondary, tertiary, and quaternary structures, we have applied Synchrotron small-angle X-ray diffraction and Fourier transform infrared spectroscopy. Upon heating a beta-Lg solution at pH 7.0, the radius of gyration Rg first decreases, indicating a partial dissociation of the dimer into the monomers, the secondary structures remaining essentially unchanged. Above 50 degrees C, the infrared spectroscopy data reveal a decrease in intramolecular beta-sheet and alpha-helical structures, whereas the contribution of disordered structures increases. Within the temperature range from 50 to 60 degrees C, the appearance of the pair distance distribution function is not altered significantly, whereas the amount of defined secondary structures declines approximately by 10%. Above 60 degrees C the aggregation process of 1% beta-Lg solutions is clearly detectable by the increase in Rg and intermolecular beta-sheet content. The irreversible aggregation is due to intermolecular S-H/S-S interchange reactions and hydrophobic interactions. Upon pressurization at room temperature, the equilibrium between monomers and dimers is also shifted and dissociation of dimers is induced. At pressures of approximately 1300 bar, the amount of beta-sheet and alpha-helical structures decreases and the content of disordered structures increases, indicating the beginning unfolding of the protein which enables aggregation. Contrary to the thermal denaturation process, intermolecular beta-sheet formation is of less importance in pressure-induced protein aggregation and gelation. The spatial extent of the resulting protein clusters is time- and concentration-dependent. The aggregation of a 1% (w/w) solution of A, B, and the mixture AB results in the formation of at least octameric units as can be deduced from the radius of gyration of about 36 A. No differences in the pressure stability of the different genetic variants of beta-Lg are detectable in our FT-IR and SAXS experiments. Even application of higher pressures (up to 10 kbar) does not result in complete unfolding of all beta-Lg variants.

Amino Acid Substitution↗
PubMedDate not supplied

Fibrillar beta-lactoglobulin gels: Part 3. Dynamic mechanical characterization of solvent-induced systems.

Oscillatory shear rheometry has been used to study the gelation of beta-lactoglobulin at ambient in 50% v/v trifluoroethanol (TFE)/pH 7 aqueous buffer and in 50% v/v ethanol (EtOH)/water at pH 2. In contrast to what was found on heating aqueous solutions at pH 2 (Part 2 of this series), a more expected "chemical gelation"-like profile was found with modulus components G' and G' ' crossing over as the gels formed and then with G' ' passing through a maximum. In addition, for the EtOH system, there was a significant modulus increase at long time, suggestive of a more complex two-step aggregation scheme. Modulus-concentration relationships were obtained for both systems by extrapolating cure data to infinite time. For the TFE gels, this data was accurately described by classical branching theory, although it could also be approximated by a constant power--law relationship. Only the latter described the modulus--concentration data for the gels in ethanol, but there were problems here of greater frequency dependence of the modulus values and much less certain extrapolation. Gel times for the TFE systems showed higher power laws in the concentration than could be explained by the branching theory in its simplest form being similar, in this respect, to the heat-set systems at pH 2. Such power laws were harder to establish for the EtOH gels as for these there was evidence of gel time divergence close to a critical concentration. Reduced G'/G'inf versus t/tgel data were difficult to interpret for the gels in ethanol, but for the TFE system they were consistent with previous results for the heat-set gels and approximated master curve superposition. The frequency and temperature dependences of the final gel moduli were also studied. In general, the networks induced by alcohols appeared more flexible than those obtained by heating.

Amyloid↗
PubMed2006

Thermodynamics of binding interactions between bovine beta-lactoglobulin A and the antihypertensive peptide beta-Lg f142-148.

The binding capacity of bovine beta-lactoglobulin variant A (beta-Lg A) for six peptides derived from beta-Lg was evaluated using an ultrafiltration method under the following conditions: pH 6.8, 40 degrees C, and a beta-Lg A/peptide molar ratio of 1:5. Only peptides beta-Lg f102-105, f142-148, and f69-83 bound in significant amounts to beta-Lg A corresponding to 1.5, 1.1, and 0.7 mol of peptide per mole of beta-Lg A, respectively. The interaction between beta-Lg A and the antihypertensive peptide beta-Lg f142-148 was investigated further by isothermal titration calorimetry. The binding isotherms at pH 6.8 and 25 degrees C confirmed that beta-Lg f142-148 bound to beta-Lg A and that the interaction followed a sequential three-site binding model with constants of association of 2 x 10(3), 1 x 10(3), and 0.4 x 10(3) M(-1) for the first, second, and third binding sites, respectively. The enthalpy of binding was exothermic for the first and second binding sites and endothermic for the third binding site. Binding of the peptide to all three sites was spontaneous as shown by the negative free energy values. These results show for the first time that beta-Lg A can bind bioactive peptides. This potential could be exploited to transport bioactive peptides and protect them in the gastrointestinal tract following their oral administration as nutraceuticals.

Animals↗
PubMed2006

Influence of pH and ionic strength on formation and stability of emulsions containing oil droplets coated by beta-lactoglobulin-alginate interfaces.

Emulsions of 0.1 wt % corn oil-in-water containing oil droplets coated by beta-lactoglobulin (0.009 wt % beta-Lg, 5 mM phosphate buffer, pH 7.0) were prepared in the absence and presence of sodium alginate (0 or 0.004 wt %). The pH (3-7) and ionic strength (0-250 mM NaCl) of these emulsions were adjusted, and the particle charge, particle size, and creaming stability were measured. Alginate adsorbed to the beta-Lg-coated droplets from pH 3 to 6, which was attributed to electrostatic attraction between the anionic polymer and cationic patches on the droplet surfaces. Droplets coated by beta-Lg-alginate had better stability to flocculation than those coated by beta-Lg alone, especially around the isoelectric point of the adsorbed proteins and at low ionic strengths (< 100 mM NaCl). At pH 5, alginate molecules desorbed from the droplet surfaces at high salt concentrations due to weakening of the electrostatic attraction.

Alginates↗
PubMed2006

Effect of the cluster size on the micro phase separation in mixtures of beta-lactoglobulin clusters and kappa-carrageenan.

The phase separation of globular protein clusters formed by heat-denatured beta-lactoglobulin (beta-lg) in mixtures with the polysaccharide kappa-carrageenan (kappa-car) has been studied at pH 7 and 20 degrees C. The effect of the protein cluster size on the phase separation was investigated by preparing clusters with radii between 20 nm and 1 mum. The formation of protein rich microdomains led to an increase of the turbidity starting at a minimum kappa-car concentration that decreased with increasing cluster size, but was only weakly dependent on the protein concentration. The size and number of microdomains do not depend much on the cluster size, but their density decreases with increasing cluster size leading to a lower turbidity.

Carrageenan↗
PubMed2006

Swelling behavior and controlled release of theophylline and sulfamethoxazole drugs in beta-lactoglobulin protein gels obtained by phase separation in water/ethanol mixture.

Physically cross-linked beta-lactoglobulin (BLG) protein gels containing theophylline and sulfamethoxazole low molecular weight drugs were prepared in 50% ethanol solution at pH 8 and two protein concentrations (6 and 7% (w/v)). Swelling behavior of cylindrical gels showed that, irrespective of the hydrated or dehydrated state of the gel, the rate of swelling was the highest in water. When the gels were exposed to water, they first showed a swelling phase in which their weight increased 3 and 30 times for hydrated and dehydrated gels, respectively, due to absorption of water, followed by a dissolution phase. The absorption of solvent was however considerably reduced when the gels were exposed to aqueous buffer solutions. The release behavior of both theophylline and sulfamethoxazole drugs from BLG gels was achieved in a time window ranging from 6 to 24 h. The drug release depended mainly on the solubility of the drugs and the physical state of the gel (hydrated or dry form). Analysis of drug release profiles using the model of Peppas showed that diffusion through hydrated gels was governed by a Fickian process whereas diffusion through dehydrated gels was governed partly by the swelling capacities of the gel but also by the structural rearrangements inside the network occurring during dehydration step. By a judicious selection of protein concentration, hydrated or dehydrated gel state, drug release may be modulated to be engineered suitable for pharmaceutical as well as cosmetics and food applications.

Ethanol↗
PubMed2006

Enzymatic cross-linking of beta-lactoglobulin: conformational properties using FTIR spectroscopy.

In this study, we use FTIR spectroscopy to probe the conformational changes of beta-lactoglobulin (beta-LG)-the main constituent of whey proteins-as subjected to enzymatic cross-linking by transglutaminase. We investigate both the amide I region (1600-1700 cm(-1)) and the C-H stretching region (2800-3100 cm(-1)). In the amide I region, spectra of denatured conformations of beta-LG, known to be necessary for cross-linking, differ according to the denaturation procedure, i.e., chemical or thermal treatment. Denaturation by chemical denaturants, dithiothreitol (DTT) or beta-mercaptoethanol, show no effect on the alpha-helix, while shifting the monomer dimer equilibrium toward higher monomer concentration. On the other hand, denaturing by thermal treatment dissociates the beta-sheets in the native structure, leading to new intermolecular beta-sheets being formed. Preheated then enzyme cross-linked beta-LG molecules show very similar spectra in the amide I region to the molecules with no cross-linking, indicating minimal effects of the cross-links on the carbonyl stretching mode. However, chemically denatured (using beta-mercaptoethanol) then enzyme cross-linked beta-LG molecules show noticeable diminution in the alpha-helix band and formation of strong hydrogen-bonded intermolecular beta-sheets. In the C-H stretching region, preheated then enzyme cross-linked beta-LG molecules exhibit a different degree of exposure of aliphatic amino acids due to the enzyme action. The same behavior is observed for DTT-treated then enzyme cross-linked beta-LG molecules. Generally, the changes in the C-H stretching region clearly indicate that hydrophobic interactions are altered upon enzymatic cross-linking.

Amides↗
PubMed2006

Strong impact of ionic strength on the kinetics of fibrilar aggregation of bovine beta-lactoglobulin.

We investigate the effect of ionic strength on the kinetics of heat-induced fibrilar aggregation of bovine beta-lactoglobulin at pH 2.0. Using in situ light scattering we find an apparent critical protein concentration below which there is no significant fibril formation for all ionic strengths studied. This is an independent confirmation of our previous observation of an apparent critical concentration for 13 mM ionic strength by proton NMR spectroscopy. It is also the first report of such a critical concentration for the higher ionic strengths. The critical concentration decreases with increasing ionic strength. Below the critical concentration mainly "dead-end" species that cannot aggregate anymore are formed. We prove that for the lowest ionic strength this species consists of irreversibly denatured protein. Atomic force microscopy studies of the morphology of the fibrils formed at different ionic strengths show shorter and curvier fibrils at higher ionic strength. The fibril length distribution changes non-monotonically with increasing ionic strength. At all ionic strengths studied, the fibrils had similar thicknesses of about 3.5 nm and a periodic structure with a period of about 25 nm.

Animals↗